Bladder cancer (BlCa) in the US is estimated to account for ~74,000 new cases of cancer and ~16,000 deaths in 2015. Despite research efforts, treatment options for muscle invasive cancers have remained largely unchanged for the past 30 years. BlCa is characterized by a gender disparity where men are three times more likely to develop BlCa than women. The theory that the androgen receptor (AR) and androgen signaling is involved in the etiology and progression of this disease is supported by numerous studies showing that AR signaling has a role in this malignancy. Our preliminary studies revealed that BlCa cells express varying levels of full length and low molecular weight (LMW) AR isoforms. LMW AR isoforms missing the ligand binding domain (LBD) are constitutively active and our studies found that cells expressing such variants are refractory to inhibition by AR antagonists, but sensitive to siRNA (against N-terminal AR) mediated AR depletion. The decreased cell viability following AR depletion is due to an increase in apoptosis and expression of the cell cycle inhibitor p27Kip1. The LMW AR has several origins, but in multiple prostate cancers is due to the expression of AR splice variants that are missing the LBD. An analysis of AR splice variants most commonly expressed in prostate tumors shows that the V7 and V1 variants are also expressed in some bladder cancer cells. siRNA-mediated depletion of the V7 variant reduces cell viability. Additionally, using 3'RACE studies we have identified a novel AR splice isoform which is expressed in two cell lines and two of five primary tumors. Treatment of cells expressing abundant LMW AR levels with the AR N- terminal domain targeting inhibitor EPI-001 effectively attenuates cell viability at a dose comparable to what was observed for prostate tumor cells. These results lead to the hypothesis that full length and LMW AR isoforms direct distinct transcriptional programs in BlCa cells and ablation of AR signaling impairs BlCa cell survival. The proposed studies have three specific aims that will test this hypothesis and define how the AR isoforms contributes to bladder tumorigenesis. The goals of Specific Aim 1 are to further characterize the novel AR isoform, and assess the levels of the LMW AR in bladder tumor and control bladder tissue, in Patient Derived Xenografts (PDX) and in bladder cancer derived cell lines. This analysis will provide a broad overview of the prevalence of AR isoforms in bladder cancer cells. Studies in Specific Aim 2 will test the hypothesis that specific AR isoforms have distinct effects on cell viability and total AR ablation effectivity inhibits cell and tumor growth. In vivo isoform specific sh-RNA-mediated ablation will determine if targeting AR isoforms inhibits tumor growth. Treatment with EPI-001 will establish if this drug is a putative BlCa therapeutic. In Specific Aim 3 RNA-seq studies will test the hypothesis that the AR isoforms regulate a defined cohort of transcripts in BlCa-derived cells. The proposed studies are expected to define the importance of the LMW AR in bladder tumorigenesis and lead to the development of new strategies for the effective treatment of this malignancy.